Abstract
The recombinant rat branched-chain alpha-ketoacid dehydrogenase kinase has been amplified from rat kidney cDNA, based on the previously reported rat cDNA sequence (Popov, K. M., Zhao, Y., Shimomura, Y., Kuntz, M. J., and Harris, R. A. (1992) J. Biol. Chem. 267, 13127-13130). This kinase was expressed in Escherichia coli as a fusion protein with bacterial maltose-binding protein (MBP). Expression was improved by overexpression of chaperonins GroEL and GroES. The MBP-kinase, when reconstituted with lipoylated recombinant E2 (dihydrolipoyl transacylase), catalyzed phosphorylation of recombinant E1 (branched-chain alpha-ketoacid decarboxylase) with a kcat of 28.5 nmol of phosphate/min/nmol of MBP-kinase at 25 degrees C. Recombinant MBP-kinase alone demonstrated a slow rate of autophosphorylation with a kcat of 3.25 pmol of phosphate/min/nmol of kinase at 25 degrees C. Serine 22 of the kinase was identified as the possible site of autophosphorylation by Edman microsequencing analysis. Autophosphorylated kinase cannot transfer phosphate to E1, indicating that autophosphorylation of kinase is not an intermediate in ATP-dependent phosphorylation of E1. Therefore, despite the reported sequence similarity to prokaryotic histidine protein kinases, the mitochondrial rat branched-chain alpha-ketoacid dehydrogenase kinase apparently does not phosphorylate E1 via a histidine-mediated phosphotransfer reaction. Significant corrections to the published cDNA sequence of rat branched-chain alpha-ketoacid dehydrogenase kinase are included.
Highlights
The recombinant rat branched-chain a-ketoacid dehydrogenase kinase has been amplified from rat kidney cDNA, based on the previously reported rat cDNA sequence
This kinase was expressed in Escherichia coli as a fusion protein with bacterial maltose-binding protein (MBP)
The branched-chain o-ketoacid dehydrogenase complex is organized around the E2 cubic core (Mr = 1.1 x 106 ), to which multiple copies of E1, E3, the kinase, and the phosphatase are attached through ionic interactions [1, 2]
Summary
El, branched-chain o-ketoacid decarboxylase; E2, dihydrolipoyl transacylase; CNBr, cyanogen bromide; EDC, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride; MBP, maltose-binding protein; MES, 2-(N-morpholino)ethanesulfonic acid; PCR, polymerase chain reaction; PVDF, polyvinylidenedifluoride; PAGE, polyacrylamide gel electrophoresis; TEV, tobacco etch virus; DTT, dithiothreitol. The purified MBPkinase was shown to possess high kinase activity when reconstituted with the lipoylated recombinant E2 In this communication, we report results obtained with the recombinant rat branched-chaino-ketoacid dehydrogenase kinase
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